Formation of mature mRNAs in higher eukaryotes requires that several processing steps occur in the nucleus prior to transport of the mRNA to the cytoplasm. For intron-containing transcripts, these steps include 5'-cap formation, methylation, 3'-end cleavage and polyadenylation, and splicing. Intronless transcripts do not undergo splicing but, in most cases, still need to undergo these other steps in pre-mRNA processing. Much information has been obtained concerning the biochemistry and machinery involved in these nuclear processing events (see Green, Annu. Rev. Cell. Biol. 7:559-599, 1991, for review). Nevertheless, the relationship of these events to nuclear export and cytoplasmic accumulation remains poorly understood.
The first evidence linking splicing and the accumulation of mRNA in the cytoplasm came from studies with SV40 (Gruss, et al., Proc. Natl. Acad. Sci. USA 76:4317-4321, 1979). Cells transfected with SV40 mutants lacking an excisable intron in the late region of the viral genome were found to synthesize late transcripts, but not to accumulate late SV40 mRNA in the cytoplasm. The requirement of an intron for efficient cytoplasmic accumulation of mRNA (i.e., intron-dependent gene expression) has been subsequently demonstrated for many other genes as well, including those encoding .beta.-globin (Hamer and Leder, Cell 17:737-747, 1979; Buchman and Berg, Mol. Cell. Biol. 8:4395-4405, 1988; Ryu, Processing of transcripts made from intron-containing and intronless protein-coding genes, Ph.D. Thesis, University of Wisconsin-Madison, Madison, Wis., 1989; Collis, et al., EMBO. J. 9:233-240, 1990), ribosomal protein L32 (Chung and Perry, Mol. Cell. Biol. 9:2075-2082, 1989), purine nucleoside phosphorylase (PNP) (Jonsson, et al., Nucleic Acids Res. 20:3191-3198, 1992), immunoglobulin .mu. (Neuberger and Williams, Nucleic Acids Res. 16:6713-6724, 1988), mouse thymidylate synthase (Deng, et al., Mol. Cell. Biol. 9:4079-4082, 1989), mouse DHFR (Gasser, et al., Proc. Natl. Acad. Sci. USA 79:6522-6526, 1982), plant alcohol dehydrogenase-1 (Callis, et al., Genes & Dev. 1:1183-1200, 1987), and triosephosphate isomerase (TPI) (Nesic, et al., Mol. Cell. Biol. 13:3359-3369, 1993). It has been proposed that the presence of introns can protect pre-mRNAs from degradation in the nucleus (Hamer and Leder, 1979, supra; Buchman and Berg, 1988, supra; Ryu and Mertz, J. Virol. 63:4386-4394, 1989), facilitate polyadenylation (Collis, et al., 1990, supra; Huang and Gorman, Nucleic Acids Res. 18:937-947, 1990; Niwa, et al., Genes & Dev. 4:1552-1559, 1990; Pandey, et al., Nucleic Acids Res. 18:3161-3170, 1990; Nesic, et al., 1993, supra; Ryu, et al., manuscript in preparation, 1995), facilitate excision of an adjacent intron (Ryu, 1989, supra; Nesic and Maquat, Genes & Dev. 8:363-375, 1994), and target mRNAs for export to the cytoplasm (Hamer and Leder, 1979, supra; Buchman and Berg, 1988, supra; Chang and Sharp, Cell 59:789-795, 1989; Legrain and Rosbash, Cell 57:573-583, 1989; Ryu and Mertz, 1989, supra).
Because of this intron requirement, the complementary DNA (cDNA) version of most genes is expressed quite poorly in mammalian cells. This poor expression cannot be overcome by use of a strong transcriptional promoter because the defects in the expression of intron-dependent genes are post-transcriptional in nature. Genomic versions of genes frequently cannot be used because (i) they have yet to be isolated, or (ii) they are too large to incorporate into useful expression vectors.
Many workers have tried to improve the expression of the cDNA versions of genes by inserting an intron back into either the protein-coding region of the gene or its 3' untranslated region. This approach is frequently unsuccessful as well because many introns (i) cannot enable efficient processing and cytoplasmic accumulation of pre-mRNAs (e.g., Ryu, 1989, supra; Nesic, et al., 1993, supra; Jonsson, et al., 1992, supra) or (ii) lead to the production of cryptically spliced mRNAs which encode incorrect proteins (Huang, et al., Mol. Cell. Biol. 10:1805-1810, 1990; Evans, et al., Gene. 84:135-142, 1989).
Interestingly, although most pre-mRNAs in higher eukaryotes require introns for efficient mRNA biogenesis, this intron requirement is not universal. The genes encoding herpes simplex virus type 1 thymidine kinase (HSV-TK) (McKnight, Nucleic Acids Res. 8:5949-5964, 1980), histone proteins (Kedes, Annu. Rev. Biochem. 48:837-870, 1978), interferon-.alpha. (Nagata, et al., Nature 287:401-408, 1980), .beta.-adrenergic receptor (Koilka, et al., Nature 329:75-79, 1987), and c-jun (Hattori, et al., Proc. Natl. Acad. Sci. USA 9148-9152, 1988) are among those genes discovered to be naturally intronless yet expressed at functional levels in higher eukaryotes.
To begin to understand the mechanism of intron-independent mRNA biogenesis, Greenspan and Weissman (Mol. Cell. Biol. 5:1894-1900, 1985), Buchman and Berg (1988, supra), and Ryu (1989, supra) constructed plasmids in which an intron plus some adjacent exon sequence from an intron-requiring .beta.-globin gene was placed 3' of the intronless sequence that encodes HSV-TK. Greenspan and Weissman (1985, supra) found that much of the resulting chimeric TK-globin RNA was polyadenylated and transported to the cytoplasm without intron excision. All three laboratories showed that the chimeric RNAs efficiently accumulated in mammalian cells regardless of whether an intron was present in the primary transcript.
One hypothesis to explain these data is that transcripts synthesized from .beta.-globin and other intron-dependent genes contain negative, cis-acting RNA sequence elements that prevent them from being properly processed and/or transported in the absence of introns; transcripts synthesized from intron-independent genes lack these negative elements and, thus, do not require introns for proper processing and transport. During the past decade, considerable data has accumulated in the literature in support of this hypothesis. For example, Legrain and Rosbash (1989, supra) found that mutations in splicing signals that converted an intron-containing gene into an intronless one led to efficient cytoplasmic accumulation of the intronless transcripts in yeast. Thus, they hypothesized that intronless transcripts are transported to the cytoplasm by default pathways.
An alternative, non-mutually exclusive hypothesis is that transcripts synthesized from intron-independent genes contain positive, cis-acting RNA sequence elements that enable them to be processed and transported regardless of whether or not introns are present. Greenspan and Weissman (1985, supra) and Buchman and Berg (1988, supra) found that various non-overlapping regions of the HSV-TK gene accumulate in cells in the absence of intron excision. Thus, HSV-TK transcripts are processed and transported to the cytoplasm regardless of introns because they either (i) lack a negative cis-acting element, or (ii) contain multiple, positive, cis-acting elements. Their data could not distinguish between these two hypotheses and was more supportive of the first hypothesis.
We show below that positive, cis-acting elements, called pre-mRNA processing enhancers (PPEs), exist. These sequence elements are capable of enabling intron-independent gene expression. Thus, their incorporation into genes provides an alternative approach to inserting introns for obtaining efficient processing and cytoplasmic accumulation of RNAs in higher eukaryotes.